Bio-Rad Aurum™ Total RNA Mini Kit - Manual (Page 21)

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D2. Transfer up to 20 mg hard animal tissue, up to 40 mg soft animal tissue,
or up to 60 mg plant tissue into an RNase-free 2.0 ml capped
microcentrifuge tube (provided). Let the tissue thaw before adding the
lysis solution or precipitation may occur.
Note: Plant tissue requires the Aurum lysis solution to be supplemented
with 2% (w/v) polyvinylpyrrolidone-40 (PVP). For each column processed,
add 14 µl PVP to every 700 µl of lysis solution before proceeding to step 3.
D3. Add 700 µl of lysis solution to the tube. Disrupt the sample by pipetting
up and down. Use a rotor-stator homogenizer for 30–60 sec or other
equivalent method to disrupt the sample.
D4. Centrifuge the lysate for 3 min and transfer the supernatant into a new
2.0 ml capped microcentrifuge tube (provided).
D5. Add 700 µl of 70% ethanol for plant tissue or 60% ethanol for animal
tissue to the supernatant. Mix thoroughly by pipetting up and down or by
using a rotor-stator homogenizer. Make sure that no bilayer is visible.
All Starting Sample Types
1. Insert an RNA binding column into a 2 ml capless wash tube (provided).
2. Decant or pipet the homogenized lysate into the RNA binding column.
Centrifuge for 30 sec (for cultured, bacterial, or yeast cells) or 60 sec (for
animal or plant tissue). Remove the RNA binding column from the wash
tube, discard the filtrate from the wash tube, and replace the column into
the same wash tube.
3. The low stringency wash solution is provided as a 5x concentrate. Add
4 volumes (80 ml) of 95–100% ethanol to the low stringency
wash solution concentrate before initial use.
4. Add 700 µl of low stringency wash solution to the RNA binding column.
Centrifuge for 30 sec. Discard the low stringency wash solution from the
wash tube and replace the column into the same wash tube.
5. The RNase-free DNase I is provided as a lyophilized powder. Reconstitute
the DNase I by adding 250 µl 10 mM Tris, pH 7.5 (not provided) to the vial
and pipetting up and down briefly to mix.
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